Cyclic AMP suppresses TGF-β-mediated adaptive Tregs differentiation through inhibiting the activation of ERK and JNK

Cellular Immunology 285 (2013) 42–48

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Cyclic AMP suppresses TGF-b-mediated adaptive Tregs differentiation through inhibiting the activation of ERK and JNK Junxia Cao 1, Xueying Zhang 1, Qingyang Wang, Xiaoqian Wang, Jianfeng Jin, Ting Zhu, Dalin Zhang, Wendie Wang, Xinying Li, Yan Li, Beifen Shen, Jiyan Zhang ⇑ Department of Molecular Immunology, Institute of Basic Medical Sciences, 27 Taiping Road, Beijing 100850, PR China

a r t i c l e

i n f o

Article history: Received 26 March 2013 Accepted 27 August 2013 Available online 6 September 2013 Keywords: cAMP TGF-b Adaptive Treg MAPKs

a b s t r a c t The second messenger cAMP is involved in the regulation of many cellular activities partially through modulating the MAPK pathways. The role of cAMP in TGF-b-mediated adaptive Tregs differentiation remains elusive. In this work, we show that cAMP inhibits antigen-nonspecific proliferation of murine CD4+ T cells without significant promotion of apoptosis. Moreover, cAMP suppresses TGF-b-induced expression of forkhead transcription factor Foxp3. 6-MB-cAMP, a site-selective activator of PKA, mimics the role of cAMP in TGF-b-induced Foxp3 expression. Further exploration reveals that TGF-b activates ERK and JNK, but not p38. cAMP and 6-MB-cAMP block TGF-b-induced activation of ERK and JNK through transcription-independent manner and transcription-dependent manner, respectively. Since direct inhibition of ERK or JNK activity mimics the effects of cAMP during this process, our work suggests that cAMP suppresses TGF-b-mediated adaptive Tregs differentiation through, at least partially, inhibiting the activation of ERK and JNK. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction T regulatory cells (Tregs)2 play a crucial role in the maintenance of immunological tolerance [1–3]. Tregs were first identified as CD4+ CD25+ T cells. Then, a forkhead transcription factor family member Foxp3 was identified as the master regulator for the development and function of CD4+ CD25+ Tregs [1–3]. Naturally derived Treg cells are generated in the thymus, whereas adaptive Tregs are generated in secondary lymphoid organs and tissues [1–3]. Antigenic or antiCD3 stimulation of CD4+ CD25 conventional T cells in the presence of cytokine TGF-b can lead to Foxp3 expression and the acquisition of suppressor function in peripheral conventional T cells [4,5]. The Smad proteins (homologs of mothers against decapentaplegic, Drosophila) are a class of proteins that function as intracellular signaling effectors for the TGF-b superfamily [4,5]. The classic Smad signaling pathways have been demonstrated essential for adaptive Tregs differentiation [4,5]. Besides, TGF-b can activate Smad-independent ⇑ Corresponding author. Fax: +86 10 68159436. E-mail address: [email protected] (J. Zhang). The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. 2 Abbreviations used: AC, adenylyl cyclases; BrdU, bromodeoxyuridine; cAMP-GEFs, cAMP-regulated guanine nucleotide exchange factors; c-FLIPL, the long form of cellular FLICE-inhibitory protein; CTX, cholera toxin; DLC, dynein light chain; FSK, forskolin; ISO, isoproterenol; MAP2K, MAP kinase kinase; MAP3K, MAPK kinase kinase; MKP-1, MAPK phosphatase-1; PGE2, prostaglandin E2; PKA, protein kinase A; Tregs, T regulatory cells. 1

0008-8749/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2013.08.006

pathways, such as MAPKs, in T cells [5–8]. There are three major groups of MAPKs in mammalian cells: ERK, JNK, and p38 [9,10]. Activation of MAPKs is typically mediated by sequential protein phosphorylation, namely, MAPK kinase kinase (MAK3K) ? MAPK kinase (MAP2K) ? MAPK [9,10]. The ERK and/or JNK pathways, but not the p38 pathway, have been shown to be involved in adaptive Tregs differentiation [5–8]. The second messenger cAMP is produced from ATP by adenylyl cyclases (AC) and can be degraded to 50 -AMP by phosphodiesterases [11,12]. AC is stimulated by a variety of physiologically relevant extracellular stimuli, such as Prostaglandin E2 (PGE2), through G-protein (Gs)-coupled membrane receptors [12]. In addition, AC can be activated by pharmacological agents, such as forskolin (FSK), which is a direct activator of AC [13], isoproterenol (ISO), which is a synthetic agonist for the b-adrenergic family of receptors [14], or cholera toxin (CTX), which causes constitutive activation of Gs by stimulating ADP-ribosylation of its a-subunit [15]. Protein kinase A (PKA) is the most important effector of cAMP action, although other cAMP-binding proteins, such as cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs), can mediate some of the biological functions of cAMP in a PKA-independent manner [16]. Activated PKA translocates into the nucleus to phosphorylate transcription factors, such as CREB at Ser133 [17]. The phosphorylation of CREB potentiates its transcription activity via recruitment of several transcription coactivators, such as CBP and p300 [17], thereby stimulating expression of target genes that are involved in many cellular activities.

J. Cao et al. / Cellular Immunology 285 (2013) 42–48

The crosstalk between the cAMP signaling pathway and the MAPK pathways has been explored. Elevation of cAMP can inhibit the upstream components of the ERK pathway in a PKA-dependent, but CREB-independent manner [18]. The inhibition of either JNK or p38 by cAMP depends on CREB-mediated transcription and involves upstream MAP2K [19,20]. However, the major effectors of cAMP-mediated inhibition of JNK or p38 activation are different. The induction of dynein light chain (DLC) is required for cAMPmediated inhibition of p38 activation, whereas the induction of the long form of cellular FLICE-inhibitory protein (c-FLIPL) and MAPK phosphatase-1 (MKP-1) is required for cAMP-mediated inhibition of JNK activation [19,20]. These observations suggest that the inhibition of ERK, JNK or p38 by cAMP could be uncoupled in certain cell context [18,21]. Since cAMP can inhibit the activation of MAPKs in T cells [22] and MAPKs contribute to TGF-b-mediated adaptive Tregs differentiation [5–8], it is possible that cAMP has a role in this process. In this work, we report that cAMP suppresses TGF-b-mediated Foxp3 up-regulation through inhibition of ERK and JNK. 2. Materials and methods

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CD28 (2 lg/ml). Proliferation was assessed by flow-cytometric analysis of CFSE dilution.

2.5. BrdU incorporation assays Cells (1  105 per well) were seeded in 96-well plates. Stimulation was affected by antibodies against CD3 (precoated, 5 lg/ml) and CD28 (2 lg/ml). 48 h later, BrdU (10 lM) was added to culture medium for 18 h. After washing, cells were resuspended in 500 ll ice-cold 0.15 M NaCl. While mixing gently, 1200 ll ice-cold 95% ethanol was added drop-wise. Cells were incubated at 4 °C in the dark for 30 min before they were washed in PBS and were treated subsequently with 2 M HCl/0.5% TritonX-100 for 30 min at room temperature. After washing in washing buffer (0.1% BSA/PBS), cells were neutralized in 0.1 M sodium borate (pH8.5) for 2 min followed by washing in washing buffer. Cells were incubated subsequently with FITC-conjugated anti-BrdU antibody in the presence of 1mg/ml RNase for 1 hr at room temperature or overnight at 4°C. Finally, cells were washed in washing buffer three times followed by flow cytometry analysis.

2.1. Mice 2.6. Apoptosis analysis Female C57 BL/6 mice at the age of 6–8 weeks were purchased from Institutes of Experimental Animals, Academy of Chinese Medical Sciences. All mice were maintained under specific pathogenfree conditions. All experiments were performed in accordance with institutional guidelines for animal care. 2.2. Reagents Forskolin, isoproterenol, cholera toxin, PGE2, PMA, ionomycin, actinomycin D, DNase, and bromodeoxyuridine (BrdU) were from Sigma–Aldrich (St. Louis, MO). 8-pCPT-20 -O-Me-cAMP and 6-MBcAMP were from Biolog (Hayward CA). Antibodies against phospho-CREB, phospho-ERK, phospho-JNK, phospho-p38, and c-FLIPL were from Cell Signaling Technology (Beverly, MA). Antibodies against MKP-1 and b-actin were from Santa Cruz Biotechnology (Santa Cruz, CA). IL-2 and TGF-b were from R&D Systems (Minneapolis, MN). CD4+ T cell isolation kit II, CD25 microbeads, and MS columns were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). CFSE was from Invitrogen (Carlsbad, CA). Fluorescence-conjugated antibodies, antibodies against CD3 and CD28, brefeldin A solution, and a fixation/permeabilization kit were from eBioscience (San Diego, CA). ECL chemiluminescence kit was from Amersham (Arlington Heights, IL). 2.3. Isolation of T cell subsets Erythrocytes were depleted by hypotonic lysis. To isolate CD4+ CD25 splenic cells, CD4+ T cells were first isolated (negative selection) from single-cell suspensions of spleens by using CD4+ T cell isolation kit II. Then the cells were incubated with CD25 microbeads. All CD25+ cells were depleted with a MS column. Purity of the cell separation was verified by immunostaining and FACS analysis. Cell purity was verified to be at least 95%. 2.4. CFSE dilution assays Cells were labeled by incubation at 106/ml in RPMI 1640 with 0.1 lM CFSE at 37 °C for 20 min, washed and resuspended in complete culture medium, as described previously [23]. Then cells (1  105 per well) were seeded in 96-well plates. Stimulation was affected by antibodies against CD3 (precoated, 5 lg/ml) and

Dual staining with FITC-conjugated Annexin V and PI was carried out to detect the induction of apoptotic cell death. Cells were washed with PBS and resuspended in 200 ll of HEPES buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2) containing 1 lg/ml Annexin V-FITC and 5 lg/ml PI (Annexin V/PI staining kit, BD Biosciences Pharmingen, San Diego, CA, USA). Following incubation for 15 min at room temperature, cells were analyzed by flow cytometry.

2.7. Adaptive Tregs differentiation Cells were stimulated with anti-CD3 (precoated, 5 lg/ml), antiCD28 (2 lg/ml), anti-IL-4 (1 lg/ml), anti-IFNc (1 lg/ml), and IL-2 (10 U/ml) in the presence or absence of TGF-b (10 ng/ml) for 96 h.

2.8. Immunoblotting analysis Whole cell lysates were prepared as previously described [24] and were resolved by SDS–PAGE before being transferred to nitrocellulose membranes. The membranes were then probed with various primary antibodies followed by peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using an ECL chemiluminescence kit.

2.9. Flow cytometry Erythrocytes were depleted by hypotonic lysis. The white cells were washed with FACS washing buffer (2% FBS, 0.1% NaN3 in PBS) twice and were then incubated with specific antibodies for 30 min on ice in the presence of 2.4G2 mAb to block FccR binding. Isotype antibodies were included as negative controls. For Foxp3 staining, after surface staining, cells were fixed and permeabilized using a fixation/permeabilization kit and stained with PE-anti-Foxp3 in accordance with the manufacturer’s instructions. Flow cytometry was performed on a Becton Dickinson FACSCalibur machine. 5  104 cells were analyzed for each sample and CD4+ T cells were gated for analysis.

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J. Cao et al. / Cellular Immunology 285 (2013) 42–48

A

B

C

Fig. 1. cAMP inhibits antigen-nonspecific CD4+ T cell proliferation without significant promotion of apoptosis. After pretreatment with or without CTX or FSK for 60 min, proliferation of CFSE-labeled (B) or CFSE-unlabeled (A,C) splenic CD4+ CD25 T cells was affected by antibodies against CD3 and CD28. 48 h later, the cells were subjected to BrdU incorporation assays (A) data are shown as mean ± SD, flow cytometry analysis of CFSE dilution (B) percentage of each generation is shown, or apoptosis analysis (C).

Foxp3

A

CD3/CD28 2.94±2.13

CD3/CD28 + TGF-β FSK 1 μM

Ctrl 46.63 ±1.54

11.76±1.29

CTX 0.1 μg/ml 10.51±0.72

CD25

Foxp3

B CD3/CD28 4.88

Ctrl 47.57

PGE2 2 μM 9.89

CD3/CD28 + TGF-β PGE2 20 μM 6.02

ISO 5 μg/ml 40.72

ISO 50 μg/ml 1.20

CD25 CD25 Fig. 2. cAMP dampens TGF-b-induced Foxp3 up-regulation. Purified splenic CD4+ CD25 T cells were subjected to the treatment with anti-CD3/CD28 antibodies plus TGF-b after pre-incubation with or without cAMP elevation agents. Then, after 4 days, the cells were assayed for CD25 and Foxp3 expression by flow cytometry. (A) Effects of forskolin or cholera toxin on TGF-b-induced Foxp3 up-regulation. Data are shown as mean ± SD. (B) Effects of PGE2 or isoproterenol on TGF-b-induced Foxp3 up-regulation.

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J. Cao et al. / Cellular Immunology 285 (2013) 42–48

+

PKI

+

+

FSK

5.3

1.6

IB

CD3/CD28 + TGF-β CD3/CD28

FSK 1μM

Ctrl

3.41±1.20

45.51±3.27

FSK 1μM+PKI 100μM

10.72±1.49

15.03±1.76

P-CREB 1

IB

B Foxp3

A

Actin CD25

C

6-MB-cAMP 0

8-pCPT-2’-O-Me-cAMP

15 30 60 120 180 0 15 30 60 120 180

Time (min)

IB

P-CREB 1.0 1.8

3.4 4.8 6.5 8.5

fold

1.0 0.7 0.6 0.9 1.1 1.4

IB

Actin

D Foxp3

CD3/CD28 + TGF-β CD3/CD28

6-MB-cAMP

Ctrl

1.89± ±0.54

45.31±4.96

8-pCPT-2’-O-Me-cAMP 43.37±3.82

7.24±3.56

CD25 Fig. 3. PKA mediates the inhibition by cAMP of TGF-b-induced Foxp3 up-regulation. (A) Purified splenic CD4+ CD25 T cells were pretreated with the specific PKA inhibitor MyPKI (100 lM) for 30 min, followed by treatment with or without FSK (1 lM, 30 min). The phosphorylation of CREB at Ser133 (P-CREB) and expression of actin were analyzed by immunoblotting (IB). Densitometric readings are shown for P-CREB and normalized with actin protein. (B) Purified splenic CD4+ CD25 T cells were preincubated with or without MyPKI (100 lM, 30 min) prior to FSK treatment (1 lM, 30 min). After induction with anti-CD3/28 antibodies plus TGF-b for 4 days, the cells were assayed for CD25 and Foxp3 expression by flow cytometry. Data are shown as mean ± SD. (C) Purified splenic CD4+ CD25 T cells were treated with 6-MB-cAMP (50 lM) or 8pCPT-20 -O-Me-cAMP (50 lM) for various periods of time. The phosphorylation of CREB and expression of actin were analyzed by immunoblotting. Densitometric readings are shown for P-CREB and normalized with actin protein. (D) Purified splenic CD4+ CD25 T cells were subjected to the treatment with anti-CD3/CD28 antibodies plus TGF-b after pre-incubation with 6-MB-cAMP (50 lM) or 8-pCPT-20 -O-Me-cAMP (50 lM). Then, after 4 days, the cells were assayed for CD25 and Foxp3 expression by flow cytometry. Data are shown as mean ± SD.

3. Results 3.1. cAMP inhibits antigen-nonspecific CD4+ T cell proliferation without significant promotion of apoptosis cAMP has been reported to inhibit antigen-specific proliferation of CD4+ T cells [25]. First, we tried to verify the proliferation inhibitory effects of cAMP. BrdU is a thymidine analog that incorporates DNA of dividing cells during the S-phase of the cell cycle. As expected, the antigen-nonspecific (CD3/CD28) CD4+ T cell proliferative response led to BrdU incorporation, which was significantly inhibited by cAMP elevation agent forskolin in a dose-dependent manner (Fig. 1A). The proliferation inhibitory effects of cAMP elevation agent cholera toxin as well as forskolin were further confirmed by CFSE dilution assays (Fig. 1B). On the other hand, apoptosis analysis revealed that the elevation of cAMP only led to slightly increased percentages of apoptosis (Fig. 1C). Thus, cAMP inhibits antigen-nonspecific CD4+ T cell proliferation without significant promotion of apoptosis.

A

+

+

TGF-β

+

FSK

IB

B

+

+

P-ERK 1.0 2.3 0.6

IB

fold

24

7

+

6-MB

+

+

8-pCPT

96

35

fold

P-ERK 1

77

37

fold

P-JNK

IB P-p38

IB 1.0 1.0 0.8

IB

fold

1.0

IB

TGF-β

+

IB

P-JNK 1

+

+

3.3 1.5

3.8 2.0

fold

Actin

Actin

Fig. 4. cAMP and 6-MB-cAMP inhibit TGF-b-induced activation of ERK and JNK. Purified splenic CD4+ CD25 T cells were activated with anti-CD3/CD28 Abs with or without TGF-b for 30 min after pre-incubation with or without FSK (1 lM), 6-MBcAMP (50 lM) and/or 8-pCPT-20 -O-Me-cAMP (50 lM). The dual phosphorylation of ERK (P-ERK), JNK (P-JNK) and p38 (P-p38) as well as expression of actin were analyzed by immunoblotting. Densitometric readings are shown for P-ERK, P-JNK, P-p38 and normalized with actin protein.

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A

+ +

+

+

+

+

+

TGF-β

+

FSK

+

P-ERK

IB 1.0 8.7

fold

6.2 0.4 3.1 1.8

P-JNK

IB 1.0 6.2

fold

1.3 2.1 4.1 4.1

IB

B

Actin

FSK (1 μM) 0

analysis showed that 6-MB-cAMP treatment led to CREB phosphorylation, whereas 8-pCPT-20 -O-Me-cAMP showed no effect on CREB phosphorylation (Fig. 3C). 6-MB-cAMP inhibits TGF-b-induced Foxp3 up-regulation to a similar extent to cAMP elevation agents, whereas 8-pCPT-20 -O-Me-cAMP exhibited no effect (Fig. 3D). Taken together, these data suggest that the inhibition by cAMP on TGF-b-induced Foxp3 up-regulation is likely mediated by PKA rather than cAMP-GEFs.

ActD

3.4. cAMP and 6-MB-cAMP inhibit TGF-b-induced activation of ERK and JNK

CTX (0.1 μg/ml)

15 30

60 120 180

0

15

30

60 120 180

3.0

7.9 7.1 2.1

1.0

2.5

3.3

4.3

IB

Time (min) P-CREB

1.0

6.7

6.2

6.9

fold

c-FLIPL

IB 1.0

1.6

1.5

1.4

1.5 1.3

1.0

1.1

1.9

1.5

1.1

0.7

fold

MKP-1

IB 1.0

2.1

1.6

2.1 2.2

1.5

1.0

0.9

1.7

1.6 1.5

1.1

IB

fold

Actin

Fig. 5. cAMP inhibits TGF-b-induced activation of ERK and JNK through distinct mechanisms. (A) Purified splenic CD4+ CD25 T cells were treated with or without actinomycin D (ActD) (1 lg/ml, 30 min) prior to FSK treatment (0.1 lM, 30 min), followed by stimulation with anti-CD3/CD28 Abs with or without TGF-b for 30 min. The dual phosphorylation of ERK and JNK as well as expression of actin was analyzed by immunoblotting. Densitometric readings are shown for P-ERK, P-JNK and normalized with actin protein. (B) Purified splenic CD4+ CD25 T cells were treated with FSK or CTX for various periods of time. The phosphorylation of CREB and expression of c-FLIPL, MKP-1, and actin were analyzed by immunoblotting. Densitometric readings are shown for P-CREB, c-FLIPL, MKP-1 and normalized with actin protein.

3.2. cAMP dampens TGF-b-induced Foxp3 up-regulation Next, we set out to analyze whether cAMP affects adaptive Tregs differentiation. For this purpose, purified splenic CD4+ CD25 T cells were subjected to anti-CD3/CD28 antibodies plus TGF-b stimulation after pretreatment with or without cAMP elevation agent cholera toxin or forskolin. Then, after 4 days, the cells were assayed for Foxp3 expression by flow cytometry. As shown in Fig. 2A, the addition of cholera toxin or forskolin reduced the percentage of CD25+ Foxp3+ cells while increasing the percentage of CD25 Foxp3 cells. Similar results were obtained when cells were pretreated with physiologically relevant cAMP inducer PGE2 or another G-protein (Gs)-coupled membrane receptor agonist isoproterenol (Fig. 2B). Taken together, these data suggest that cAMP might inhibit adaptive Tregs differentiation. 3.3. PKA mediates the inhibition by cAMP of TGF-b-induced Foxp3 upregulation PKA is the most important effector of cAMP action. To explore the involvement of PKA in the inhibition by cAMP of TGF-b-induced Foxp3 up-regulation, we employed MyPKI, a specific cell-permeable PKA inhibitor [26]. Forskolin treatment led to the phosphorylation of CREB at Ser133, which implies the activation of PKA (Fig. 3A). At the dose that MyPKI partially inhibited forskolin-induced CREB phosphorylation (Fig. 3A), MyPKI partially reversed forskolin-mediated inhibition on TGF-b-induced Foxp3 expression (Fig. 3B), suggesting that PKA mediates the inhibition by cAMP of TGF-b-induced Foxp3 up-regulation. To further address this issue, 8-pCPT-20 -O-Me-cAMP, a potent specific activator of cAMP-GEFs, and 6-MB-cAMP, a site-selective activator of PKA but a poor activator of cAMP-GEFs, were used [27]. Immunoblotting

Next, we set out to explore the possible involvement of MAPKs in the regulation of adaptive Treg differentiation by cAMP. In this scenario, splenic CD4+ CD25 T cells were activated with anti-CD3/CD28 antibodies with or without TGF-b for 30 min. Immunoblotting analysis revealed that TGF-b enhanced the dual phosphorylation of ERK and JNK, which is required for the activation of ERK and JNK [19–20] (Fig. 4A). Under the same conditions, TGF-b showed no role on p38 phosphorylation (Fig. 4A). As expected, the addition of forskolin significantly inhibited TGF-b-mediated activation of ERK and JNK, but showed no role on p38 phosphorylation (Fig. 4A). Moreover, 6-MB-cAMP, which mimics the role of cAMP in TGF-b-induced Foxp3 expression, also exerted an inhibitory role on TGF-b-mediated activation of ERK and JNK (Fig. 4B). 3.5. cAMP inhibits TGF-b-induced activation of ERK and JNK through distinct mechanisms Our previous studies have shown that cAMP can inhibit JNK and/or p38 activation through the PKA-CREB pathway [19–20]. cAMP can also inhibit upstream components of the ERK pathway in a PKA-dependent, but CREB-independent manner [18]. It is of interest how cAMP might inhibit TGF-b-induced activation of ERK and JNK during adaptive Treg differentiation. For this purpose, CD4+ T cells were pretreated with or without forskolin, followed by treatment with TGF-b in the presence or absence of the RNA synthesis inhibitor actinomycin D, or left untreated. The inhibition by forskolin of TGF-b-induced JNK phosphorylation was abolished by actinomycin D (Fig. 5A). However, forskolin still significantly inhibits TGF-b-induced ERK phosphorylation under the same conditions (Fig. 5A). Furthermore, Immunoblotting analysis revealed that both forskolin and cholera toxin activated CREB, which was correlated with the up-regulation c-FLIPL and MKP-1 in CD4+ T cells (Fig. 5B). These data suggest that the cAMP might also inhibit JNK activation in CD4+ T cells through CREB-mediated up-regulation of c-FLIPL and MKP-1 whereas the inhibition by cAMP of TGF-b-induced ERK activation is transcription-independent. 3.6. The inhibition of ERK or JNK activation mimics the effects of cAMP in TGF-b-mediated adaptive Tregs differentiation Then, the role of ERK, JNK, or p38 in TGF-b-mediated adaptive Tregs differentiation was analyzed by adding specific inhibitors into the culture. Flow cytometry revealed that the addition of either a JNK or ERK inhibitor partially attenuated Foxp3 expression in TGF-b-primed CD4+ cells (Fig. 6A). Conversely, the addition of a p38 inhibitor did not alter Foxp3 expression in TGF-b-primed CD4+ cells (Fig. 6A). Consistently, CD4+ CD25 T cells from JNK1/ mice exhibited impaired Foxp3 expression 4 days after anti-CD3/ CD28 antibodies plus TGF-b stimulation (Fig. 6B). Moreover, the addition of an ERK inhibitor showed an additive effect on the inhibition of TGF-b-induced adaptive Tregs differentiation (Fig. 6B). Taken together, these data suggest that the suppression by cAMP of ERK and JNK activation contributes to, at least partially, the inhibition by cAMP of TGF-b-induced adaptive Tregs differentiation.

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A Foxp3

CD3/CD28 + TGF-β CD3/CD28

Ctrl

2.81± ±0.43

JNK inhibitor

43.61±1.21

ERK inhibitor

33.81±1.92

28.03±3.96

p38 inhibitor 46.14±0.74

CD25

B

WT +

JNK1-/+ +

+ +

+ +

CD3/CD28 TGF-β ERK inhibitor

Foxp3

+ 1.69±0.56

42.40±3.03

32.60±1.77

5.11±6.48

CD25 Fig. 6. The inhibition of ERK or JNK activation mimics the effects of cAMP in TGF-b-mediated adaptive Tregs differentiation. Purified splenic CD4+ CD25 T cells were subjected to the induction with anti-CD3/28 antibodies plus TGF-b in the presence or absence of MAPKs inhibitors (10 lM each). After 4 days, the cells were assayed for CD25 and Foxp3 expression by flow cytometry. (A) Purified splenic CD4+ CD25 T cells from wild type (WT) mice were used. Data are shown as mean ± SD. (B) Purified splenic CD4+ CD25 T cells from wild type mice or JNK1/ mice were used. Data are shown as mean ± SD.

cAMP

TGF-β

TGF-β

PKA c-FLIPL

MKK4/7

JNK

Nucleus

MEK1/2 PKA ERK

MKP-1

CREB c-flipL

Adaptive Tregs differentiation

mkp-1

Adaptive Tregs differentiation

Fig. 7. A schematic presentation of the molecular mechanism by which cAMP inhibits TGF-b-mediated adaptive Tregs differentiation. Elevation of cAMP can inhibit the upstream components of the ERK pathway in a PKA-dependent, but CREB-independent manner. The inhibition of JNK by cAMP depends on CREBmediated induction of c-FLIPL and MKP-1. MKP-1 directly inhibits JNK whereas cFLIPL targets MKK7. Both JNK and ERK contribute to TGF-b-mediated adaptive Tregs differentiation. Therefore, cAMP suppresses TGF-b-mediated Foxp3 up-regulation through, at least partially, inhibition of ERK and JNK.

4. Discussion MAPKs are key regulators of numerous cellular events and their activity is tightly controlled by other intracellular signaling pathways, such as the cAMP pathway. Our data show that cAMP, most likely through PKA, inhibits TGF-b-induced activation of ERK and JNK. The ERK and/or JNK pathways, but not the p38 pathway, have been shown to be involved in adaptive Tregs differentiation [5–8]. Consistent with the previous reports, we have found that the inhibition of ERK or JNK activation mimics the effects of cAMP in TGF-b-mediated adaptive Tregs differentiation. Therefore, cAMP suppresses adaptive Tregs differentiation through, at least partially, inhibiting the activation of ERK and JNK. The previous observations suggest that cAMP inhibits ERK in a PKA-dependent, but CREB-independent manner [18]. On the other

hand, the inhibition of JNK activation by cAMP depends on CREBmediated transcription [20]. Indeed, this work shows that the inhibition by forskolin of TGF-b-induced JNK phosphorylation was abolished by actinomycin D. However, forskolin still significantly inhibits TGF-b-induced ERK phosphorylation under the same conditions. Accordingly, cAMP induces the up-regulation of c-FLIPL and MKP-1, two inhibitors of the JNK pathway. Thus, cAMP inhibits TGF-b-induced activation of ERK and JNK through distinct mechanisms in CD4+ T cells (Fig. 7). In addition, it is noticed that actinomycin D treatment appears to directly inhibit TGF-b-induced activation of ERK and JNK even though it has distinct effects on the basal levels of ERK and JNK phosphorylation (Fig. 5A and data not shown). Thus, it is possible that a component in TGF-b signaling, which is essential for ERK and JNK activation, depends on de novo protein synthesis to maintain its protein levels. Future studies are required to address this issue. The role of cAMP in the induction of Foxp3 expression seems to be context-dependent. cAMP elevation agents PGE2 and vasoactive intestinal peptide have been reported to induce Foxp3 expression and T regulatory cell function in human CD4+ T cells [28,29]. Our findings are not consistent with these previous reports. The discrepancy might be due to the role of TGF-b. Our work uses exogenous TGF-b to induce adaptive Tregs differentiation whereas no exogenous TGF-b was added into the co-culture system of human CD4+ T cells. Another key factor might be the species. Our study focuses on murine CD4+ T cells whereas previous reports focus on human CD4+ T cells. Moreover, cAMP elevation agents and cellintrinsic cAMP might exert distinct roles in Foxp3 induction since it has been shown that the specific deficiency of the stimulatory Ga subunit of Gs (Gas) in T cells exhibited no role in adaptive Tregs differentiation [30]. The mechanisms underlying the discrepancy remain to be explored. Chronic psychological stress is associated with persistent activation of the hypothalamic–pituitary–adrenal (HPA) axis, which leads to continuously elevated levels of stress hormones such as

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glucocorticoid and catecholamines [31,32]. Catecholamines activate AC. Therefore, many cell types respond to catecholamines with an increase in cAMP levels [33]. Because chronic psychological stress has long been reported to trigger the onset of autoimmune disorders [34,35], it is possible the inhibition by cAMP elevation agents of TGF-b-mediated adaptive Tregs differentiation contributes to the progression of autoimmunity. The lack of in vivo models hinders the clarification of the role of catecholamines-induced cAMP elevation in this progress. Another limitation of the reported observations here is that we can’t compare the importance of the MAPK pathway versus the classic Smad signaling pathways during TGF-b-mediated adaptive Tregs differentiation. Disclosures The authors have no financial conflict of interest to disclose. Acknowledgments We thank Qianqian Dai and Fengjun Xiao for technical assistance. This work was supported by grants from National Natural Science Foundation of China (31270960, 31100544), the National Key Technologies R & D Program for New Drugs (2013ZX09103003-010), and National Key Basic Research Program of China (2010CB911904). References [1] K.H. Mills, Regulatory T cells: friend or foe in immunity to infection?, Nat Rev. Immunol. 4 (2004) 841–855. [2] J.D. Fontenot, M.A. Gavin, A.Y. Rudensky, Foxp3 programs the development and function of CD4+ CD25+ regulatory T cells, Nat. Immunol. 4 (2003) 330–336. [3] S. Hori, T. Nomura, S. Sakaguchi, Control of regulatory T cell development by the transcription factor Foxp3, Science 299 (2003) 1057–1061. [4] M. Long, S.G. Park, I. Strickland, M.S. Hayden, S. Ghosh, Nuclear factor-kappaB modulates regulatory T cell development by directly regulating expression of Foxp3 transcription factor, Immunity 31 (2009) 921–931. [5] L. Lu, J. Wang, F. Zhang, Y. Chai, D. Brand, X. Wang, D.A. Horwitz, W. Shi, S.G. Zheng, Role of SMAD and non-SMAD signals in the development of Th17 and regulatory T cells, J. Immunol. 184 (2010) 4295–4306. [6] Y.E. Zhang, Non-Smad pathways in TGF-beta signaling, Cell Res. 19 (2009) 128–139. [7] M.E. Kalland, N.G. Oberprieler, T. Vang, K. Taskén, K.M. Torgersen, T cellsignaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells, J. Immunol. 187 (2011) 5233–5245. [8] L. Xu, A. Kitani, C. Stuelten, G. McGrady, I. Fuss, W. Strober, Positive and negative transcriptional regulation of the Foxp3 gene is mediated by access and binding of the Smad3 protein to enhancer I, Immunity 33 (2010) 313–325. [9] J. Wang, R. Tang, M. Lv, Q. Wang, X. Zhang, Y. Guo, H. Chang, C. Qiao, H. Xiao, X. Li, Y. Li, B. Shen, J. Zhang, Defective anchoring of JNK1 in the cytoplasm by MKK7 in Jurkat cells is associated with resistance to Fas-mediated apoptosis, Mol. Biol. Cell 22 (2011) 117–127. [10] Y. Guo, W. Wang, J. Wang, J. Feng, Q. Wang, J. Jin, M. Lv, X. Li, Y. Li, Y. Ma, B. Shen, J. Zhang, Receptor for activated C kinase 1 promotes hepatocellular carcinoma growth by enhancing mitogen-activated protein kinase kinase 7 activity, Hepatology 57 (2013) 140–151. [11] M.D. Houslay, D.R. Adams, PDE4 cAMP phosphodiesterases: modular enzymes that orchestrate signalling cross-talk, desensitization and compartmentalization, Biochem. J. 370 (2003) 1–18. [12] R.K. Sunahara, R. Taussig, Isoforms of mammalian adenylyl cyclase: multiplicities of signaling, Mol. Interv. 2 (2002) 168–184.

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